In the United States and elsewhere in the technologically developed world it is customary and often legally mandated to assess the capacity of consumer products such as cosmetics, detergents, or other materials likely to be handled by the general public, to cause temporary or permanent damage to the human skin. It is clearly desirable and necessary to provide adequate labeling for corrosive skin irritants and even to restrict commercialization of products which contain components extremely damaging to the skin. It is estimated that more than 2 million tests are performed annually in the United States to examine the various cosmetic and household products for their potential harmfulness to the human skin. As in any instance where large numbers of tests need to be performed to screen for a particular property, it is desirable to have a testing procedure that is rapid, inexpensive, and reliable. An in vitro test would be most appropriate.
Such an in vitro test is not currently available for substances that might irritate the skin. The most commonly used screening procedure is the Draize rabbit skin test (Draize, J.H., et al., J Pharmacol Exp Therap (1944) 82:377). The disadvantages of this test are legion. (See for example, Weil, C.S., and Scala, R.A., Toxicol Appl Pharmacol (1971) 19:276-360; Phillips, L., et al., Toxicol Appl Pharmacol (1972) 21:369-382.) First, as an in vivo procedure, it involves some degree of maltreatment of test animals and considerable expense. Second, it is lengthy. The procedure consists of placing the substance to be tested onto a small square of gauze and applying this to the clipped trunk of the rabbit. After 24 hours the degree of irritation is scored subjectively.
Many attempts have been made to refine and improve this method using different exposure conditions, exposure times, and different animal models. (See for example, Roudabush, R.L., et al., Toxicol Appl Pharmacol (1965) 7:559; Wooding, W.M., and Opdyke, D.L., J Soc Cosmet Chem (1967) 18:809; Kligmann, A.M., and Wooding, W.M., J Invest Dermatol (1967) 49:78-94; Marzulli, F.N., Toxicol Appl Pharmacol (1965) 7:Suppl.2:79-85.) However, the inherent deficiencies of this in vivo testing approach make impossible the attainment of an inexpensive, rapid, reproducible, and predictive test for predicting skin irritation properties.
Although as in all animal tests, the correlation with effects of the materials in human subjects is imperfect, the Draize skin test has become the standard and any new in vitro tests should produce results similar to those of Draize. Thus correlation with Draize skin results can be used to evaluate the effectiveness of such a new test.
The clear desirability of an in vitro procedure has led a number of researchers to devise tests involving cell and organ cultures as opposed to whole animals. For example, the method of Benoit, J., et al., Toxic in Vitro (1987) 1:91, employs cultured human fibroblasts and uses the ability of a material to produce a cytotoxic effect as a measure of its skin irritation potential. Cytotoxic effects in cell culture as a measure of skin irritation has also been studied by Borenfreund, E., and Borrero, 0., Cell Biol Toxicol (1984) 1:55-65; Choman, B.R., J Invest Dermat (1984)40:177-182; and Scaife, M.C., Fd Chem Toxic (1985) 23:253-258. Helman, R.G., et al., Fund and Appl Toxicol (1984) 7:94-100, measured the leakage of cellular enzymes in cultured discs of mouse skin to evaluate the potential skin irritation of various chemicals. An additional method employs an isolated perfused skin flap for skin irritation measurements and was developed by Edmond, J., et al., Fund and Appl Toxicol (1986) 7:444-453.
These foregoing methods require either living cells in tissue culture or living organs in tissue culture. These methods suffer from lack of reproducibility, lack of objectivity, and lack of significant correlation with the Draize method.
Several other in vitro methods which do not use living cells have been developed to study skin irritation by surfactants. Some of these methods evaluate biophysical properties of the stratum corneum such as its swelling, and its permeability. Other methodologies determine the denaturation of proteins as indicators of potential skin irritation by surfactants. Blake-Haskins, J.C., et al., J Soc Cosmet Chem (1986) 37:199-210 related the irritancy of a surfactant to its ability to cause collagen matrices to swell and hold water. Ernst, R., J Am Chem Soc (1980) 57:93, measured the change in the activity of enzymes (i.e., denaturation) and related the decreased activity to the potential skin irritation of surfactants. While this method produced correlative results for some surfactants, many irritating solvents and preservatives did not alter the enzyme activity. The skin roughness caused by anionic surfactants was found to be related to their ability to denature protein materials (Imokawa, G., et al., J Am Oil Chem Soc (1975) 52:175). This method produced correlative results for anionic surfactants but not for cationic and nonionic surfactants and therefore, has not been shown to be universal in predicting skin irritation properties of surfactants. Finally, Kligman et al., J Soc Cosmet Chem (1988) 39:267, have shown that the pH of a solution of bovine serum albumin treated with a surfactant correlated with skin irritation. However, this relationship held for anionic surfactants but not for nonionic or cationic surfactants.
These methods fail to correctly mimic the simple three compartment model, which is the art-recognized model of skin irritation. Compartment one, the outer barrier or stratum corneum, which is a network of fibrous keratin and collagen molecules has a role to protect the epidermis and dermis from chemical assault and to provide the correct hydration for the skin. A change in this layer, such as the swelling of the stratum corneum, represents only the perturbation of this one compartment. Compartment two, the epidermis with its cells and intracellular components, the outer barrier. The third compartment, the dermis, lies beneath the epidermis and provides the vascularization and metabolic activity to nourish compartments one and two and is seldom involved in irritation except for extremely corrosive substances which destroy the stratum corneum and the epidermis. Skin irritation, registered as erythema and edema, requires changes in the structure and organization of macromolecules typical of compartments one and/or two when challenged with chemical irritants.
While the in vitro procedures above provide an alternative to the strictly in vivo approach of the Draize test, they do not achieve the simplicity and standardization that one expects from a new in vitro test. The method of the present invention offers such a test. It provides a quick, standard, reproducible, and objective measure of the capacity for any material to cause irritation in the human skin. It does not involve the use of animals and does not require the expense of maintaining, caging, and feeding them.